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Home , Alirocumab, Inclisiran

Intervention to improve LDL receptor function Emerging

Marina Cuchel, MD, PhD Perelman School of Medicine University of Pennsylvania Most Lipid Lowering Drugs Affect LDL-C Levels By Up-Regulating the LDLR

B100

apoB MTP VLDL TG TG bile acid sequestrants IDL PCSK9 -inhibitors LDL TG Cuchel NLA 2017 Do we really need more LDL-C lowering drugs?

• LDL-C still not a goal with existing LLTs in a significant portion of subjects • Drug intolerance (statins) • Cost of new treatments • Other reasons

Cuchel NLA 2017 Outline

• PCSK9 – beyond monoclonal antibodies • ETC-1002 – inhibiting upstream HMGCoAR • Replacing LDLR - Gene • ANGPTL3 inhibitors and other drug affecting LDL production

Cuchel NLA 2017 PCSK9 enhances LDLR degradation

Cuchel NLA 2017 Use of PCSK9i is complementary to that of statins

PCSK9i evolucumab [other monoclonal ABs] statins

HMG-CoA

↑LDLR ↑PCSK9 ↓LDL-C ↓Cholesterol

Cuchel NLA 2017 PCSK9 inhibition is very effective in lowering LDL-C

Sabatine MS et al. N Engl J Med 2017. DOI: 10.1056/NEJMoa1615664 Cuchel NLA 2017 PCSK9 – beyond monoclonal Antibodies

• Monoclonal PCSK9i are expensive • Require high doses • Are injectable • ?May lose responsiveness over time?

Cuchel NLA 2017 Targeting PCSK9 with novel strategies

• Vaccines • siRNA • Small molecules

Cuchel NLA 2017 Targeting PCSK9 with novel strategies Vaccines

antibodies response

SREBP-2 pathway

↑ HMGCoA Red ↑ LDL-R Target protein ↑Epitope PCSK9 identification Conjugation with Administration Antibodies foreign peptide production

Clinicaltrials.gov NCT02508896 Cuchel NLA 2017 Vaccine against PCSK9 lowers LDL-C in primates

Crossey E. Vaccine, 2015 , 33, 5747–5755 Cuchel NLA 2017 Targeting PCSK9 with novel strategies siRNA

SREBP-2 pathway

↑ HMGCoA Red ↑ LDL-R ↓PCSK9 Gene silencing Clinicaltrials.gov NCT02597127, NCT02314442 Cuchel NLA 2017 The RNAi inhibitor of PCSK9 - - lowers PCSK9 and LDL-C levels in

Ray KK et al. N Engl J Med 2017;376:1430-1440. Cuchel NLA 2017 Targeting PCSK9 with novel strategies Small molecules

Adnectins Binding Proteins to PCSK9

SREBP-2 pathway

↑ HMGCoA Red ↑ LDL-R ↑ PCSK9

Cuchel NLA 2017 Targeting PCSK9 with novel strategies Small molecules

Blocking TGN transport and/or autocleavage

SREBP-2 pathway

↑ HMGCoA Red ↑ LDL-R ↑ PCSK9

Cuchel NLA 2017 Outline

• PCSK9 – beyond monoclonal antibodies • ETC-1002 – inhibiting upstream HMGCoAR • Replacing LDLR - Gene therapy • ANGPTL3 inhibition

Cuchel NLA 2017 Inhibiting upstream HMGCoA Reductase ETC-1002 ()

Citrate

ACL Acetyl-CoA

FA HMG-CoA statins TG HMG-R ↓LDL-C ↓Cholesterol

Cuchel NLA 2017 Inhibiting upstream HMGCoA Reductase ETC-1002 (Bempedoic Acid)

ETC-1002 Acetyl-CoA Synthase Citrate ETC-1002 CoA ACL Acetyl-CoA

FA HMG-CoA statins TG HMG-R ↓LDL-C ↓Cholesterol

• 18 studies listed in Clinicaltrials.gov, 5 actively recruiting Cuchel NLA 2017 ETC-1002 lower LDL-C in patients with or without intolerance

Thompson J Clin Lipidol 2016. 10: 556-567, Cuchel NLA 2017 Outline

• PCSK9 – beyond monoclonal antibodies • ETC-1002 – inhibiting upstream HMGCoAR • Replacing LDLR - Gene therapy • ANGPTL3 inhibition

Cuchel NLA 2017 Homozygous Familial

12 Y.O. female LDL-C=780 mg/dL, xanthomas since age 3, coronary heart disease, cardiac bypass FH is Caused by Mutations in Genes Affecting LDL Receptor Functionality

B100 B100

apoB VLDL apoB VLDL TG TG TG TG X X X LDLR LDLR IDL IDL TG LDL TG LDL

HeFH HoFH LDL-C >190 mg/dl LDL-C > 500 mg/dl • The impaired LDLR functionality leads to a decreased clearance of LDL particles from plasma Cuchel NLA 2016 FH Is Most Frequently Caused by LDLR Mutations

• Mutations in LDLR are found in ~95% of the confirmed cases of HoFH • Other genes are: APOB, PCSK9, LDLRAP1 • More than 1,000 LDLR mutations have been reported • Based on the mutations patients can be divided in receptor negative (<2% activity) or receptor defective • The type of mutations affect the LDL-C levels and the response to treatment

Cuchel NLA 2017 Penn HoFH Cohort: LDLR Negative Subjects Have a More Severe Phenotype

LDLR negative (n=18) LDLR defective (n=16) p Median (range) Age (yr) at visit 1 11.5 (3.3 - 29) 28.1 (3.3 - 44.6) 0.008 Age at 1st xanthomas 2.0 (0.25 - 4) 7.0 (1 - 15) <0.001 Age (yr) at FH dx 3.0 (0.5 - 7) 8.0 (2 - 17) <0.001 TC (mg/dl) at dx 895 (602-1260) 686 (519-900) 0.002 Age (yr) at start of Rx 5.0 (1.2 - 10) 16.0 (2 - 31) 0.001 Age (yr) at CAD 12.5 (6 - 16) 22.0 (16 - 37) 0.004 Lipid profile at visit 1 Mean (SD) TC (mg/dl) 618 (238) 453 (162) 0.025 LDL-C (mg/dl) 535 (214) 393(159) 0.040 Apo B (mg/dl) 388 (142) 293 (92) 0.032

Kolansky DM, Am J Cardiol 2008; 102:14-38-43 Cuchel NLA 2017 New Lipid Lowering Drugs Affect LDL-C Levels by Inhibiting VLDL Secretion B100

apoB MTP VLDL TG TG

IDL LDL TG Cuchel NLA 2017 Would -directed gene therapy of LDLR for HoFH work?

B100

apoB MTP VLDL X TG X ? IDL LDL TG

Cuchel NLA 2017 Liver transplantation normalizes LDL-C in patients with Homozygous Familial Hypercholesterolemia

Starzl, et al., The Lancet, 1984. Cuchel NLA 2017 Gene Therapy Viruses Are Natural Gene Delivery Vehicles

• Viruses have evolved to deliver genes efficiently to cells • Viruses can be engineered to both express the gene of interest and impair their ability to replicate. These engineered viruses are often referred to as viral vectors.

Hu W-S, Pathak VK. Pharmacol Rev. 2000;52(4):493-512. Liver directed ex vivo gene therapy in patients with HoFH

Pre + statin Pre - statin Post - statin Post + statin

Grossman et al, Nature Medicine 1995 Cuchel NLA 2017 Recombinant AAV Vectors for Gene Therapy

• Adeno Associated Virus (AAV)are non-pathogenic in humans • AAV are much less immunogenic that vectors used before • Very unlikely to integrate into the host cell genome • In vivo approach is possible • Several AAVs are used in gene therapy for several genetic conditions • AAV8 can produce efficient and prolonged gene transfer in the liver Clinical Experience With AAV Gene Therapy

Examples of clinical experience with AAV vectors Disease AAV vector Route of Target Clinical outcome Latest Reported administration organ Duration of Efficacy

Lipoprotein Glybera®, alipogene Multiple Muscle Reduction in 6+ years lipase (LPL) tiparvovec, AAV1- intramuscular pancreatitis deficiency1 LPLS447X injections Hemophilia B2 AAV8-FIX Single intravenous Liver Reduction in 4+ years infusion bleeding episodes and in FIX infusions

1. Scott LJ. Drugs. 2015;75(2):175-182. 2. 2. Nathwani AC, et al. N Engl J Med. 2014;371(21):1994-2004. AAV-LDLR administration reduce LDL-C levels and atherosclerotic burden in animal models

*humanized mouse model: LDLR-Apobec-DKO/hApoB-Tg mice

Kassim SH et al Human Gene Therapy. 2013, 24: 19-26 Kassim SH et al Plos One. 2010, Cuchel NLA 2017 FHGT002 - Phase I/II in HoFH Objectives: Key inclusions criteria: 1. To determine the safety of AAV-LDLR 1. Male or female ≥ 18 years of age. administration in patients with HoFH 2. LDLR mutations at both alleles, clinical presentation consistent with HoFH. 2. To assess efficacy (% changes in LDL-C levels) 3. NAbs titer <1:10 3. To assess immune response following vector 4. Stable concurrent allowed lipid lowering administration : – statins, ezetimibe, bile acid seq., PCSK9i, apheresis

Single iv infusion

Screening stage Treatment evaluation Follow up

Weeks

0* 4 8 12* 26 52 Up to 5 yrs

* Kinetic study Secondary endpoints Safety endpoint

Phase I/II open label dose escalation (3+3 design)

2.5x1012 gc/kg n = 3 7.5x1012 gc/kg n = 3 7.5x1012 gc/kg n = up to 6

Safety review Safety review

Clinicaltrials.gov NCT02651675 Outline

• PCSK9 – beyond monoclonal antibodies • ETC-1002 – inhibiting upstream HMGCoAR • Replacing LDLR - Gene therapy • ANGPTL3 inhibitors (and other drug affecting LDL production)

Cuchel NLA 2017 Lowering LDL-C levels by decreasing LDL production mipomersen B100

apoB VLDL MTP ApoC-III LPL Acetyl-CoA TG ANGPTL3 Carboxylase TG lomitapide inhibitors ↓LDL-C gemcabene

IDL LDL TG Cuchel NLA 2017 ANGPTL3 inhibitor as a treatment for HoFH

• ANGPTL3 is a liver secreted protein that inhibits Lipoprotein Lipase and possibly Endothelial Lipase • Carriers of ANGPTL3 mutations have combined hypolipidemia LDL-C TG HDL-C

Musunuru K et al. N Engl J Med 2010;363:2220-2227. Cuchel NLA 2017 ANGPTL3 inhibitor as a treatment for HoFH

• ANGPTL3 inhibition reduces LDL in ldlr -/- mice • Can ANGPTL3 inhibition be effective in reducing LDL-C in HoFH?

Wang Y. JLR 2015. 56: 1296-1307 Cuchel NLA 2017 ANGPTL3 inhibitor as a treatment for HoFH

• Main study of REGN1500 () in HoFH subjects is completed. OLE study is ongoing (NCT02265952) • Preliminary data show that it is effective in reducing LDL-C levels in HoFH • Evinacumab received Breakthrough Therapy Designation

Cuchel NLA 2017 Lowering LDL-C levels by decreasing LDL production

inhibitors

B100 ANGPTL3 apoB MTP VLD LLPL X TG ? X TG

IDL LDL TG

Cuchel NLA 2017 Summary

• Upregulation of the LDL Receptor is a valid and proven approach to lower LDL-C levels • The LDL receptor continue to be an active drug target • Alternative approaches to block PCSK9 activity are actively pursued • ETC-1002, an inhibitor of ACL, is effective in reducing LDL-C levels • LDL receptor gene replacement using AAV vectors is being currently pursued • ANGPTL3 inhibition is a promising approach to lower LDL-C via non LDLR-mediated mechanisms.

Cuchel NLA 2017